In the USA stroke kills 155,000 people per annum and is the third largest cause of death, after diseases of the heart and cancer. Furthermore 4,000,000 stroke survivors in the USA alone are coping with its debilitating effects. Stroke rates rise sharply with age, thus the increasing aging population will further increase its incidence. There is a brief window of opportunity in the hours following stroke during which the damage to the brain can be kept to a minimum, but the design of rational therapy would be facilitated if we had greater understanding of the underlying processes. Neuronal damage following stroke is amplified by the pathological release of glutamate (excitotoxicity), the consequent chronic activation of NMDA selective glutamate receptors and the influx of calcium into the cell. Mitochondrial calcium loading and consequent dysfunction is implicated not only in stroke but also in chronic neurodegenerative disorders such as Parkinson's and Huntington's diseases. The long term goal of this study is therefore to develop a comprehensive understanding of the acute and chronic consequences for the mitochondrion and neuron of pathological NMDA receptor activation, to use this information to devise and test neuroprotective strategies for the brief therapeutic window following stroke, and to relate these findings to the slow neurodegenerative disorders in which mitochondrial dysfunction is implicated. Mitochondria generate ATP, but also detoxify reactive oxygen species produced by the respiratory chain, control the cellular redox state and regulate cytoplasmic Ca2+. Any combination of these pathways may dysfunction in these disorders and the challenge is to unravel their complex interactions to identify primary lesions and suggest therapeutic and preventative strategies. The hypothesis that this application will test is that changes in mitochondrial bioenergetics function initiated during brief glutamate exposure continue even in the absence of receptor activation and are responsible for the delayed death of the neuron. To test this hypothesis we propose studies with the following specific aims. 1. To establish the immediate and delayed bioenergetics consequences of transient pathological NMDA receptor activation for cultured cerebellar granule neurons and their in situ mitochondria. While the granule cell in vivo is relatively resistant to excitotoxicity, the extensive existing information on the bioenergetics properties of these cells, coupled with their tolerance to a range of mitochondrial inhibitors makes the preparation suitable to establish principles applicable to neurons in general. 2. To establish the mechanism by which the initial transient exposure to glutamate initiates the subsequent mitochondrial dysfunction. 3. To test hypotheses that the lethality of glutamate in this model can be affected by controlling membrane potential and respiratory chain capacity. 4. To test the hypothesis that mitochondria at specific intracellular locations may be selectively vulnerable to glutamate excitotoxicity because of their proximity to NMDA receptors, or their location in regions of high-energy demand.